Recent advances in genetic engineering have enabled the development of new biological platforms to produce molecules heretofore only synthesized by chemical routes. Although microbial fermentation is routinely exploited for the production of small molecules and proteins of industrial and/or pharmaceutical importance (antibiotics, enzymes, vaccines, etc.), the possibility of using green plants for the manufacture of high-volume chemicals and materials has become an increasingly attractive alternative.
There are two obvious advantages of using green plants to produce large amounts of compounds that are traditionally manufactured through normal chemical synthesis. First, green plants constitute a renewable energy source, as opposed to petrochemical production. Because of their unique photosynthetic capability, the only raw materials that are required to produce carbon-based compounds in green plants are carbon dioxide, water, and soil, with sunlight providing the ultimate source of energy. Second, in comparison to existing fermentation facilities which are limited in size, green plants constitute a huge available biomass that could easily accommodate the large amounts of chemicals that are required for certain high-volume, low-cost applications. However, there are still a number of important obstacles that must be overcome before green plants can be exploited for this purpose. For example, living plants might not be able to tolerate high levels of certain compounds, even if they are naturally found in plants, albeit at much lower levels. Although toxicity also poses potential problems for the production of chemicals through fermentation, plants are vastly more complex than fungi, bacteria, or other microorganisms, especially with regard to genetics, metabolism and cellular differentiation.
Fortunately, however, plants and animals deploy remarkably similar mechanisms for detoxifying the broad range of toxic compounds to which they are exposed or produce themselves (Sandermann, Pharmacogenetics 4:225-241 (1994)). In both kingdoms, the detoxification of exogenous and endogenous toxins is a three-phase process (Coleman, Trends Plant Sci. 2:144-151 (1997); Wink, M. In The Plant Vacuole: Advances in Botanical Research; Leigh, R. A., Sanders, D. and Callow, J. A., Eds.; Academic Press: London, New York, 1997; Vol. 25, pp 141-169). Phase I (activation) is the introduction or exposure of functional groups of the appropriate reactivity for phase II enzymes. Cytochrome P450-dependent monooxygenases and mixed function oxidases are examples of phase I enzymes. Phase II (conjugation) is covalent attachment of the activated compound to a bulky hydrophilic molecule that increases its water solubility and is thought to promote its recognition by phase III transporters. Phase III (elimination) is transport of the conjugates out of the cytosol into intracellular compartments and/or the extracellular space. In mammals, the conjugates are typically excreted into the urine or bile. In plants, that otherwise lack bona fide excretory organs, the conjugates are often sequestered in the vacuole, a large acidic organelle that constitutes 40-90% of the total cell volume.
Due to their pharmacological importance, the best characterized phase II reactions are probably those catalyzed by mammalian UDP-glucuronyltransferases which attach glucuronic acid to a wide range of acceptor molecules (Meech and Mackenzie, Clinical and Experimental Pharmacology and Physiology 24:907-915 (1997)). Closely related homologs exist in plants, as judged by the presence of more than one hundred ORFs in arabidopsis encoding polypeptides bearing a C-terminal consensus sequence common to all members of the UDP-glycosyltransferase superfamily (Mackenzie et al., Pharmacogenetics 7:255-269 (1997); Lim et al., J. Biol. Chem. 276:4344-4349 (2001)), but less is known about these enzymes than their mammalian counterparts. The majority of the plant enzymes are thought to use UDP-glucose as the sugar donor, but their natural substrates and physiological functions largely remain elusive, despite the increasing number of purified proteins that have been rigorously characterized over the last several years (Lim et al., supra; Jackson et al., J. Biol. Chem. 276:4350-4356 (2001); Ford et al., J. Biol. Chem. 273:9224-9233 (1998); Vogt et al., Plant J. 19:509-519 (1999); Lee and Raskin, J. Biol. Chem. 274:36637-36642 (1999); Fraissinet-Tachet et al., FEBS Lett. 437:319-323 (1998)). However, it is tacitly assumed that one of the key roles of plant UDP-glucosyltransferases is to target endogenous and exogenous toxins to the vacuole.
Most of the products of secondary metabolism in plants are glycosylated (Harborne, J. Introduction to Ecological Biochemistry, 4th ed.; Academic Press: London, 1993), as are many herbicides after modification by phase I enzymes. An impressive array of conjugated species, including coumaryl glucosides, flavonoids, anthocyanins, cardenolides, soponins, cyanogenic glucosides, glucosinolates, and betalains, are known to be stored in the vacuole (Wink, M., supra). Based on these observations and the fact that most UDP-glucosyltransferases are located in the cytosol, glucosylation has been invoked as a prerequisite for uptake and accumulation in the vacuole. In addition, in vitro experiments clearly demonstrate that isolated vacuoles and/or vacuolar membrane vesicles are able to take up certain glucose conjugates, while the parent molecules are not transported (Wink, M., supra).
p-Hydroxybenzoic acid (pHBA) is a naturally occurring plant secondary metabolite that has been shown to have a number of useful applications. It is the major monomer of Liquid: Crystal Polymers (LCPs), ˜55% of the total weight, and chemical precursor for the synthesis of methylparaben, which is a preservative that is commonly used in the food and cosmetic industries. Since it is anticipated that the global demand for pHBA will exceed one hundred million pounds per year by the end of the decade, green plants represent an attractive platform for the production of this compound.
Indeed, it has recently been shown (Siebert et al., Plant Physiol. 112:811-819 (1996)) that it is possible to increase pHBA levels in tobacco two to three orders of magnitude using a chloroplast-targeted version of E. coli chorismate pyruvate lyase (CPL). Interestingly, virtually all of the overproduced pHBA (>95%) was converted to two glucose conjugates, a phenolic glucoside with the glucose moiety attached to the aromatic hydroxyl group, and a glucose ester where the sugar is attached to the aromatic carboxyl group. Although both glucose conjugates accumulate in the vacuole, they have very different chemical properties and physiological roles.
For example, the pHBA glucose ester (like other acetal esters) is characterized by high free energy of hydrolysis, which makes it very simple to recover the parent compound with low concentrations of either acid or base. This could greatly reduce the cost of producing pHBA in plants. Furthermore, it is well established that certain glucose esters are able to serve as activated acyl donors in enzyme-mediated transesterification reactions (Li et al., Proc. Natl. Acad. U.S.A. 97, 12:6902-6907 (2000); Lehfeldt et al., Plant Cell 12, 8:1295-1306 (2000)), In light of these observations, it would be extremely desirable to control the partitioning of pHBA glucose conjugates in vivo. For example, by overexpressing an appropriate glucosyltransferase in transgenic plants that generate large amounts of pHBA, it might be possible to accumulate all of the desired compound as the glucose ester, which is easily hydrolyzed to free pHBA. While the above scenario is extremely attractive, it requires an enzyme with the appropriate properties and molecular information that would allow access to the gene (e.g., its nucleotide or primary amino acid sequence).
Several publications describe plant enzymes that catalyze the formation of glucosides and/or glucose esters of hydroxybenzoic acids. For example, Klick et al. (Phytochemistry 27(7):2177-2180 (1988)) reported that glucose conjugates of hydroxybenzoic acids are present as low abundance secondary metabolites in a wide range of plant species, and occur in nature as both glucosides and glucose esters. Gross et al. (Phytochemistry 10:2179-2183 (1983)) described an enzyme activity from oak trees that catalyzes the formation of glucose esters of hydroxybenzoic acids, including pHBA. Bechthold et al. (Archives of Biochemistry and Biophysics 288(1):39-47 (1991)) described an enzyme activity in cell cultures of Lithospermum erythrorhizon that was very specific for pHBA and only formed the pHBA phenolic glucoside. In a subsequent study (Li et al., Phytochemistry 46(1):27-32 (1997)), the same protein was purified to homogeneity and subjected to digestion with endoprotease Lys-C. Although several peptide fragments were successfully sequenced, the authors did not publish this information. Chorismate pyruvate-lyase (CPL)-mediated production of pHBA in transgenic tobacco plants resulted in accumulation of the pHBA phenolic glucoside and pHBA glucose ester (Siebert et al., Plant Physiol. 112:811-819 (1996)). Moreover, similar results were obtained when pHBA was generated in the cytosol using a different bacterial gene, namely, the HCHL (4-hydroxycinnamoyl-CoA hydratase/lyase) gene from Pseudomonas fluorescens (Mayer et al., Plant Cell 13(7):1669-1682 (2001). Li et al. (Plant Cell Physiol. 38(7):844-850 (1997)) described glucosyltransferase activities in tobacco cell cultures that catalyze the formation of both pHBA conjugates, but the experiments were performed with crude extracts, not purified proteins. None of the reports cited above describe at the molecular level any genes or proteins that are responsible for the pHBA phenolic or ester glucosides.
On the other hand, Fraissinet-Tachet et al. (FEBS Lett. 437(3):319-323(1998)) has presented the complete nucleotide sequences of two closely related UDP-glucosyltransferases from tobacco that are active with pHBA, and characterized the purified recombinant proteins. However, both enzymes interact with a wide variety of substrates that bear little resemblance to each other. Moreover, both enzymes attach glucose to the hydroxyl and carboxyl group of pHBA. Lee and Raskin (J. Biol. Chem. 274:36637-36642 (1999)) published the complete DNA sequence of a different tobacco UDP-glucosyltransferase that is also able to glucosylate pHBA. However, this protein also exhibits very broad substrate specificity and yields both glucosides and glucose esters of various hydroxybenzoic acids and hydroxycinnamic acids. Additionally, Milkowski and colleagues (Milkowski et al., Planta 211(6):883-886 (2000); Milkowski et al., FEBS Lett. 486(2):183-184 (2000)) and Lim et al., (supra) describe a family of genes from cruciferous plants, Brassica napus and Arabidopsis thaliana, that encode for UDP-glucosyltransferases that exclusively catalyze the formation of glucose esters. However, in the case of the arabidopsis homologs (Lim et al., supra), the only substrates examined were cinnamic acid derivatives, and there was tremendous variation in the substrate specificity of the different enzymes even within this class of compounds. Moreover, although pHBA was one of the test substrates for the Brassica protein (Milkowski et al., Planta 211(6):883-886 (2000)) and the arabidopsis proteins (Milkowski et al., FEBS Lett. 486(2):183-184 (2000)), the authors reported that this compound was not glucosylated under the conditions of their in vitro assay.
Three UDP-glucosyltransferase proteins from Arabidopsis thaliana that are capable of glucosylating pHBA have been reported to attach glucose exclusively to the aromatic carboxyl group to form the pHBA glucose ester (Lim et al., J. Biol. Chem. 277: 586-592 (2002)). One of these proteins, referred to as 84A1, is identical to GT 3 described in the present application, based on structural similarity and kinetic properties, but is not a member of the new subfamily of UDP-glucosyltransferases that are identified herein. Although GT3/84A1 is able to form the pHBA glucose ester, this enzyme exhibits a marked preference for hydroxycinnamic acid derivatives, like sinapic acid, and has a relatively low turnover number for pHBA. The other two arabidopsis proteins described in the above disclosure (e.g., 75B1 and 75B2) are even more distantly related to the UDP-glucosyltransferases that we have discovered. For example, both proteins are less than 45% identical to the instant Grape GT at the amino acid sequence level when compared by gap alignment. Consequently, none of these proteins (GT3/84A1, 75B1, or 75B2) are a suitable catalyst for purposes of the present invention.
The problem to be solved, therefore, is the lack of enzymes that preferentially catalyze the formation of glucose esters of pHBA and other hydroxybenzoic acid derivatives with sufficiently high turnover for use in various applications, both in vitro and in vivo.